Low Dose of BPA Induces Liver Injury through Oxidative Stress, Inflammation and Apoptosis in Long-Evans Lactating Rats and Its Perinatal Effect on Female PND6 Offspring.

Bisphenol A (BPA) is a phenolic compound used in plastics elaboration for food protection or packaging. BPA-monomers can be released into the food chain, resulting in continuous and ubiquitous low-dose human exposure. This exposure during prenatal development is especially critical and could lead to alterations in ontogeny of tissues increasing the risk of developing diseases in adulthood. The aim was to evaluate whether BPA administration (0.036 mg/kg b.w./day and 3.42 mg/kg b.w./day) to pregnant rats could induce liver injury by generating oxidative stress, inflammation and apoptosis, and whether these effects may be observed in female postnatal day-6 (PND6) offspring. Antioxidant enzymes (CAT, SOD, GR, GPx and GST), glutathione system (GSH/GSSG) and lipid-DNA damage markers (MDA, LPO, NO, 8-OHdG) were measured using colorimetric methods. Inducers of oxidative stress (HO-1d, iNOS, eNOS), inflammation (IL-1ß) and apoptosis (AIF, BAX, Bcl-2 and BCL-XL) were measured by qRT-PCR and Western blotting in liver of lactating dams and offspring. Hepatic serum markers and histology were performed. Low dose of BPA caused liver injury in lactating dams and had a perinatal effect in female PND6 offspring by increasing oxidative stress levels, triggering an inflammatory response and apoptosis pathways in the organ responsible for detoxification of this endocrine disruptor.

Activation of NLRP3 Inflammasome in Liver of Long Evans Lactating Rats and Its Perinatal Effects in the Offspring after Bisphenol F Exposure.

The liver is the organ responsible for the metabolism and detoxification of BPF, the BPA analogue that is replacing it in plastic-based products. It is not known whether BPF can trigger inflammatory responses via the NLRP3 inflammasome, which plays a major role in the development of liver disease. The aim of this study was to evaluate nitrosative stress species (RNS) and NLRP3 inflammasome activation in the liver of lactating dams after BPF exposure. Moreover, it was studied whether this effect could also be observed in the liver of female and male offspring at postnatal day 6 (PND6). 36 Long Evans rats were randomly distributed according to oral treatment into three groups: Control, BPF-low dose (LBPF; 0.0365 mg/kg b.w./day) group and BPF-high dose (HBPF; 3.65 mg/kg b.w./day) group. The levels of nitrosative stress-inducing proteins (eNOS, iNOS, HO-1d), NLRP3 inflammasome components (NLRP3, PyCARD, CASP1) and proinflammatory cytokines (IL-1ß, IL-18, IFN-γ and TNF-α) were measured by gene and protein expression in the liver of lactating dams and in female and male PND6 offspring. Lactating dams treated with LBPF showed a significant increase in iNOS and HO-1d, activation of NLRP3 components (NLRP3, PyCARD, CASP1) and promoted the release of proinflammatory cytokines such as IL-1ß, IL-18, IFN-γ and TNF-α. Similar effects were found in female and male PND6 offspring after perinatal exposure. LBPF oral administration and perinatal exposure caused an increase of nitrosative stress markers and proinflammatory cytokines. Also, NLRP3 inflammasome activation was significantly increased in in the liver of lactating dams and PND6 offspring.

Developmental effects of perinatal exposure to PBDE and PCB on gene expression in sexually dimorphic rat brain regions and female sexual behavior.

The developing nervous system is a potential target of environmental contaminants such as polybrominated diphenylethers (PBDE), which accumulate in the biosphere. We compared effects of 2,2',4,4',5-pentabromo-BDE (PBDE99), a PBDE congener present in environmental samples, and PCB on brain development. Time-pregnant rats were subcutaneously injected with PBDE99 (1 or 10mg/kg), the PCB mixture Aroclor 1254 (10mg/kg), or vehicle from gestational day 10-18. mRNA levels of genes involved in central control of reproductive functions and sexual behavior were analyzed by real time RT PCR in two sexually dimorphic brain regions, medial preoptic area (MPO) and ventromedial hypothalamus (VMH) of adult offspring of both sexes. Exposure to PBDE99 or the PCB mixture during pre- and postnatal development affected mRNA expression levels in a treatment-, region- and sex-specific manner, and changed the sensitivity of target genes to estradiol. The sex difference in progesterone receptor mRNA levels of VMH normally seen in untreated controls was abolished by both, PBDE99 and PCB. Estrous cycles were significantly affected, and preliminary experiments suggest an impairment of female sexual behavior. Our data indicate that developmental exposure to PBDE99 at doses below signs of general toxicity affects the regulation of estrogen target genes in rat brain. Since PBDE99 was detected in blood and adipose tissue of adult offspring, these effects may result from interactions with developmental processes, with adult functions, or a combination of both.

Oxidative stress increases in liver of lactating rats after BPF-low-dose exposure: perinatal effects in the offspring.

Bisphenol F (BPF) is replacing Bisphenol A (BPA) in the manufacture of products due to endocrine-disrupting effects. BPF monomers can also be released into the environment and enter the food chain, resulting in human exposure to low doses. Since bisphenols are primarily metabolized by the liver, this organ is more vulnerable to lower doses of bisphenols than others. Exposure during prenatal development may increase the risk of diseases in adulthood. The aim was to evaluate whether BPF administration could generate oxidative stress in liver of lactating rats, and whether these effects may be also observed in female and male postnatal day 6 (PND6) offspring. Long Evans rats received oral treatment: Control, BPF-low-dose (LBPF) 0.0365 mg/kg b.w./day, and BPF-high-dose (HBPF) 3.65 mg/kg b.w./day. The levels of antioxidant enzymes (CAT, SOD, GR, GPx and GST), glutathione system (GSH, GSSG) and lipid damage markers (MDA, LPO) were measured using colorimetric methods in liver of both lactating dams and in PND6 offspring. Mean values were analyzed using Prism-7. LBPF affected liver defense mechanisms (antioxidant enzymes and glutathione system), increasing ROS levels and producing lipid peroxidation in lactating dams. Similar effects were found in female and male PND6 offspring as a consequence of perinatal exposure.

Converging Effects of Three Different Endocrine Disrupters on Sox and Pou Gene Expression in Developing Rat Hippocampus: Possible Role of microRNA in Sex Differences.

Endocrine disrupting chemicals (EDCs) can impair hippocampus-dependent behaviors in rat offspring and in children. In search for key processes underlying this effect, we compared the transcriptomes of rat hippocampus on postnatal day 6 after gestational and lactational exposure to three different EDCs at doses known to impair development of learning and memory. Aroclor 1254, a commercial PCB mixture (5 mg/kg or 0.5 mg/kg), or bisphenol A (5 mg/kg or 0.5 mg/kg) were administered in chow, chlorpyrifos (3 mg/kg or 1 mg/kg) was injected subcutaneously. Male hippocampus exhibited a common effect of all three chemicals on genes involved in cell-autonomous processes, Sox6, Sox11, Pou2f2/Oct2, and Pou3f2/Brn2, all upregulated at the high dose. Additional genes of the Sox and Pou families were affected by only one or two of the chemicals. Real time RT PCR showed a comparable expression change for bisphenol A also at the lower dose. Female hippocampus exhibited much fewer genes with expression changes (almost none with false discovery rate <0.05), and none of the genes of the Sox and Pou families was affected. Since gene network analyses in male hippocampus suggested a link between Sox6 and miR-24, known to be repressed by activation of ER-alpha and to repress Sox6 in other tissues, this microRNA was measured. miR-24 was downregulated by all chemicals at the high dose in males. Values of Sox6 mRNA and miR-24 were inversely correlated in individual male hippocampus samples, supporting the hypothesis that the change in Sox6 expression resulted from an action of miR-24. In contrast, miR-24 levels remained unchanged in hippocampus of females. A sexually dimorphic response of miR-24 may thus be at the basis of the sex difference in Sox6 expression changes following exposure to the three chemicals. ER-alpha expression was also sex-dependent, but the expression changes did not parallel those of potential downstream genes such as Sox6. Sox6 is known to suppress differentiation of Parvalbumin (Pvalb)-expressing interneurons. Individual Sox6 levels (FPKM) were inversely correlated with levels of Pvalb, but not with markers of Sox6-independent interneuron subpopulations, Nos1 and 5HT3aR. Effects on interneuron development are further suggested, in males, by expression changes of Nrg1 and its receptor Erbb4, controlling interneuron migration. Our study disclosed new types of EDC-responsive morphogenetic genes, and illustrated the potential relevance of microRNAs in sexually dimorphic EDC actions.

Environmental exposure to lead, but not other neurotoxic metals, relates to core elements of ADHD in Romanian children: performance and questionnaire data.

Neurobehavioral measures of attention, and clinical features of the attention-deficit hyperactivity disorder (ADHD) have been studied in pediatric environmental lead research. However rarely, if ever, have performance measures of attention or executive functions and questionnaire-based quantitative ADHD-observations been studied in the same subjects. We examined associations between pediatric blood lead concentrations (PbB), as well as those of mercury (Hg), and aluminum (Al), and performance in four different attention tasks, as well as behavioral ratings from an ICD-10 (hyperactivity) and DSM-IV-coded (attention deficit) German questionnaire (FBB-ADHS). Asymptomatic, 8-12 year old children from two Romanian cities were studied, namely Bucharest and Pantelimon, a city near a metal-processing plant. Blood was analyzed for Pb, Al, and Hg. Data from 83 children were available for final analysis. We assessed attention performance by means of four tasks of the computer-based ADHD-taylored German KITAP-battery. We also received questionnaire ratings from parents and teachers covering three ADHD-dimensions. Multiple linear regression analysis was used to estimate associations between the three neurotoxic trace metals in blood and the different ADHD features. After adjusting for eleven potentially confounding variables we found consistent borderline to significant associations between Pb, but not other metals, in blood and various performance- and questionnaire data. False alarm responses (FAR) in the KITAP subtests rather than response latencies exhibited positive associations with PbB. Questionnaire ratings for ADHD dimensions also revealed PbB-related adversity. With any two-fold increase of PbB outcome changed markedly, namely up to 35%. Restriction to children with PbBs<10mug/dl had only a marginal influence on outcome.The converging evidence from performance- and questionnaire data confirms that core elements of ADHD are adversely affected by low environmental PbB even below 10mug/dl, but not by other neurotoxic trace metals. Observed effect-sizes are considerably larger than those typically found for lead-related IQ-deficit, thus suggesting that attention deficit could be the more basic adverse effect of lead in children. This is the first study from Central and Eastern Europe dealing with links between environmental exposure of children to neurotoxic metals and ADHD.

Region-specific growth effects in the developing rat prostate following fetal exposure to estrogenic ultraviolet filters.

BACKGROUND AND OBJECTIVES: Exposure to environmental endocrine disruptors is a potential risk factor for humans. Many of these chemicals have been shown to exhibit disruption of normal cellular and developmental processes in animal models. Ultraviolet (UV) filters used as sunscreens in cosmetics have previously been shown to exhibit estrogenic activity in in vitro and in vivo assays. We examined the effects of two UV filters, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC), in the developing prostate of the fetal rat. METHODS: Pregnant Long Evans rats were fed diets containing doses of 4-MBC and 3-BC that resulted in average daily intakes of these chemicals corresponding to the lowest observed adverse effects level (LOAEL) and the no observed adverse effects level (NOAEL) doses in prior developmental toxicity studies. Using digital photographs of serial sections from postnatal day 1 animals, we identified, contoured, and aligned the epithelial ducts from specific regions of the developing prostate, plus the accessory sex glands and calculated the total volume for each region from three-dimensional, surface-rendered models. RESULTS: Fetal exposure to 4-MBC (7.0 mg/kg body weight/day) resulted in a significant increase (p < 0.05) in tissue volume in the prostate and accessory sex glands. Treated males exhibited a 62% increase in the number of ducts in the caudal dorsal prostate. Increased distal branching morphogenesis appears to be a consequence of exposure in the ventral region, resulting in a 106% increase in ductal volume. CONCLUSIONS: 4-MBC exposure during development of the male reproductive accessory sex glands exhibited classical growth effects associated with estrogenic endocrine disruptors. The different regional responses suggest that the two developmental processes of ductal outgrowth and branching morphogenesis are affected independently by exposure to the environmental chemicals.

Developmental toxicity of UV filters and environmental exposure: a review.

Several ultraviolet (UV) filters exhibit estrogenic, some also anti-androgenic activity. They are present in waste water treatment plants, surface waters and biosphere including human milk, suggesting potential exposure during development. Developmental toxicity was studied in rats for the UV filters 4-methylbenzylidene camphor (4-MBC, 0.7, 7, 24, 47 mg/kg/day) and 3-benzylidene camphor (3-BC, 0.07, 0.24, 0.7, 2.4, 7 mg/kg/day) administered in chow to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. Neonates exhibited enhanced prostate growth after 4-MBC and altered uterine gene expression after both chemicals. 4-MBC and 3-BC delayed male puberty and affected reproductive organ weights of adult offspring. Effects on the thyroid axis were also noted. Expression and oestrogen sensitivity of oestrogen-regulated genes and nuclear receptor coregulator levels were altered at mRNA and protein levels in adult uterus, prostate and brain regions involved in gonadal control and sexual behaviour. Female sexual behaviour was impaired by both filters; 3-benzylidene camphor caused irregular cycles. Classical endpoints exhibited lowest observed adverse effect levels (LOAELs) and no observed adverse effect levels (NOAELs) of 7/0.7 mg/kg for 4-MBC and 0.24/0.07 mg/kg for 3-BC. Molecular endpoints were affected by the lowest doses studied. Our data indicate that the potential risk posed by endocrine active UV filters warrants further investigations.

Organochlorine pesticides in placenta in Kyrgyzstan and the effect on pregnancy, childbirth, and newborn health.

Organochlorine pesticides (OCPs) were determined by gas chromatography in 241 placentas from cotton-growing regions, 121 placentas from an urban area (city of Osh), and 146 placentas from unpolluted mountain regions of Kyrgyzstan. Manifestations of disease were recorded in the mothers during pregnancy and parturition and in their newborns during the first 6 days of life. OCPs were detected in 240 out of 508 placentas (47.2%), with increased incidence in the two polluted regions (65%), particularly in placentas from women living near former pesticide storehouses and agro air-strips (99%), but only in 2.7% of placentas from the unpolluted region. α-, ß-, and γ-hexachlorocyclohexane (HCH); DDT; DDE; aldrin; and heptachlor were detected. The sum of concentrations of all OCPs (total OCPs) was calculated for each of the 240 placentas with detectable OCPs (median 9.5 µg/kg placenta, mean 88.3 µg/kg, range 0.1-3070 µg/kg). The incidence of health problems in four subgroups of this data set, with increasing levels of total OCPs, was compared with the incidence of health problems in the group of 268 placentas, where OCPs were undetectable. Relative risk of health problems in both, mothers and newborns, increased significantly, in a concentration-dependent manner, with increasing levels of total OCPs (p < 0.0001). Health complications with increased incidence in OCP-exposed newborns included, i.a., low birth weight, congenital malformations, infections, and stillbirths, in OCP-exposed mothers preterm delivery, (pre-)eclampsia/gestosis, and frequency of hospitalizations after delivery (infections). Women living near former pesticide storehouses and agro airstrips should be considered as being at risk. Reduction of exposure is urgently needed.

Estrogen sensitivity of target genes and expression of nuclear receptor co-regulators in rat prostate after pre- and postnatal exposure to the ultraviolet filter 4-methylbenzylidene camphor.

BACKGROUND AND OBJECTIVES: In previous studies, we found that the ultraviolet filter 4-methyl-benzylidene camphor (4-MBC) exhibits estrogenic activity, is a preferential estrogen receptor (ER)-beta ligand, and interferes with development of female reproductive organs and brain of both sexes in rats. Here, we report effects on male development. METHODS: 4-MBC (0.7, 7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to offspring until adulthood. mRNA was determined in prostate lobes by real-time reverse transcription-polymerase chain reaction and protein was determined by Western blot analysis. RESULTS: 4-MBC delayed male puberty, decreased adult prostate weight, and slightly increased testis weight. Androgen receptor (AR), insulin-like growth factor-1 (IGF-1), ER-alpha, and ER-beta expression in prostate were altered at mRNA and protein levels, with stronger effects in dorsolateral than ventral prostate. To assess sensitivity of target genes to estrogens, offspring were castrated on postnatal day 70, injected with 17beta-estradiol (E(2); 10 or 50 microg/kg, sc) or vehicle on postnatal day 84, and sacrificed 6 hr later. Acute repression of AR and IGF-1 mRNAs by E(2), studied in ventral prostate, was reduced by 4-MBC exposure. This was accompanied by reduced co-repressor N-CoR (nuclear receptor co-repressor) protein in ventral and dorsolateral prostate, whereas steroid receptor coactivator-1 (SRC-1) protein levels were unaffected. CONCLUSIONS: Our data indicate that 4-MBC affects development of male reproductive functions and organs, with a lowest observed adverse effect level of 0.7 mg/kg. Nuclear receptor coregulators were revealed as targets for endocrine disruptors, as shown for N-CoR in prostate and SRC-1 in uterus. This may have widespread effects on gene regulation.

Salbutamol exhibits androgenic activity in vitro.

BACKGROUND: Salbutamol has been shown to mediate anabolic effects after intravenous administration. However, the mechanism responsible for the anabolic actions of salbutamol remains unknown. AIM: To investigate the potential mechanism by which salbutamol mediates anabolic effects in vitro. METHODS: The potential androgenic activity of salbutamol was investigated in vitro by the A-Screen assay that measures androgen-dependent inhibition of proliferation of the androgen receptor (AR)-positive human mammary carcinoma cell line, MCF7-AR1. RESULTS: The assay was validated with three known androgens; methyltrienolone (R1881), 5alpha-dihydrotestosterone (DHT) and danazol. IC50 values of R1881, DHT and danazol, 4.41x10(-11), 4.44x10(-11) and 1.08x10(-8) M, respectively, were in the ranges known from earlier studies. Our results demonstrate that salbutamol exhibits androgenic activity, with an IC50 value of 8.93x10(-6) M. Anti-estrogenic or cytotoxic effects, which might have interfered with the assay, were excluded by additional experiments on wild-type MCF7 and MCF7-AR1 cells, respectively. CONCLUSION: These data indicate that salbutamol exerts anabolic effects through androgen receptor agonistic activity in vitro.

Efficiency control of dietary pesticide intake reduction by human biomonitoring.

In spite of food safety controls for pesticide residues, a conventional diet still leads to a noticeable exposure of the general population to several pesticides. In a pilot study the response of exposure reduction by organic diet intervention on the urinary levels of pesticide metabolites was investigated. In the study two adult individuals were kept on a conventional diet for 11days and morning urine voids were collected at the last four days of the period. Afterwards, the participants switched to exclusively organic food intake for 18days and likewise morning urine samples were collected at the last four days of this period. In the urine samples six pyrethroid metabolites, six dialkylphosphates, four phenolic parameter for organophosphate pesticides and carbamates, 6-chloronicotinic acid (ClNA) as parameter for neonicotinoid insecticides, seven phenoxy herbicides, glyphosate and its metabolite AMPA were quantified using gas chromatographic mass spectrometric methods. Generally, the comparative analyses revealed greater shares as well as higher levels of the parameters in the samples taken during the common diet period compared to the organic diet period. Considerable decrease of the levels was found for almost all pyrethroid metabolites, dialkyphosphates and phenoxy herbicids, as well as for the phenolic metabolites 4-nitrophenol and 3,5,6-trichloropyridinol. In contrast, higher values were found for the organic diet period for ClNA and the metabolite of coumaphos in one of the volunteers. The present study confirms the results of former studies which indicated that an organic diet intervention results in considerable lower exposure to organophosphate pesticides and pyrethroids. It also verifies the former experience that monitoring of urinary parameters for non-persistent pesticides permits a reliable efficiency control of short-time effects by dietary interventions. Additionally to former studies, the results of the present study highlight the need of an extension of the parameter spectrum to all prominent pesticide groups.

Gene expression and estrogen sensitivity in rat uterus after developmental exposure to the polybrominated diphenylether PBDE 99 and PCB.

Considering the presence of polybrominated diphenylethers (PBDEs) in human milk and cord blood, and the estrogenic activity of some congeners, it is conceivable that PBDEs may interact with developing neuroendocrine systems. We investigated effects of 2,2',4,4',5-pentabromo-DE (PBDE 99), a major congener in human milk, on development of brain and reproductive organs, with focus on estrogen target gene expression. Time-pregnant Long Evans rats were subcutaneously injected with PBDE 99 (1 or 10 mg/kg/day), the PCB mixture Aroclor 1254 (10 mg/kg/day), known to interfere with sexual development, or vehicle, from gestational day (GD) 10 to GD 18. In female offspring, anogenital distance was unaffected by PBDE 99 but increased by Aroclor; puberty (vaginal opening) was not significantly changed. Adult PBDE 99-exposed offspring exhibited unchanged uterine weight but increased ovarian weight. Uterine mRNA levels of estrogen target genes were determined by real-time PCR. Progesterone receptor (PR) mRNA was down-regulated at both PBDE 99 doses, estrogen receptor alpha (ER alpha), ER beta and insulin-like growth factor-I (IGF-I) were up-regulated at the lower dose. Aroclor induced different effect patterns. In order to investigate possible changes in sensitivity of target genes to estrogen, some offspring were ovariectomized at 10 weeks of age, s.c. injected with estradiol-17beta (E2, 10 microg/kg) or vehicle at 12 weeks, and sacrificed 6 h later. PBDE 99 dose-dependently reduced the magnitude of IGF-I mRNA induction by E2, and increased the magnitude of ER beta repression. PBDE 99 also influenced baseline levels of PR, IGF-I and ER beta mRNAs in ovariectomized, vehicle-injected controls. These data indicate that developmental exposure to PBDE 99 at doses devoid of general toxicity, affects the regulation of estrogen target genes in uterus. Since PBDE 99 was detected in blood and adipose tissue of adult offspring, these effects may result from interactions with developmental processes, adult functions, or a combination of both.

Estrogen target gene regulation and coactivator expression in rat uterus after developmental exposure to the ultraviolet filter 4-methylbenzylidene camphor.

Because the estrogen receptor (ER) ligand type influences transactivation, it is important to obtain information on molecular actions of nonclassical ER agonists. UV filters from cosmetics represent new classes of endocrine active chemicals, including the preferential ER beta ligands 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor. We studied estrogen target gene expression in uterus of Long Evans rats after developmental exposure to 4-MBC (0.7, 7, 24, and 47 mg/kg x d) administered in feed to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. 4-MBC altered steady-state levels of mRNAs encoding for ER alpha, ER beta, progesterone receptor (PR), IGF-I, androgen receptor, determined by real-time RT-PCR in uterus of 12-wk-old offspring. Western-blot analyses of the same tissue homogenates indicated changes in ER alpha and PR but not ER beta proteins. To assess sensitivity to estradiol (E2), offspring were ovariectomized on d 70, injected with E2 (10 or 50 microg/kg sc) on d 84, and killed 6 h later. Acute up-regulation of PR and IGF-I and down-regulation of ER alpha and androgen receptor by E2 were dose-dependently reduced in 4-MBC-exposed rats. The reduced response to E2 was accompanied by reduced coactivator SRC-1 mRNA and protein levels. Our data indicate that developmental exposure to 4-MBC affects the regulation of estrogen target genes and the expression of nuclear receptor coregulators in uterus at mRNA and protein levels.

Sex- and region-specific alterations of progesterone receptor mRNA levels and estrogen sensitivity in rat brain following developmental exposure to the estrogenic UV filter 4-methylbenzylidene camphor.

Recently, we reported on in vitro and in vivo estrogenic activity of UV filters and on developmental toxicity of 4-methylbenzylidene (4-MBC) camphor [Schlumpf, M., Cotton, B., Conscience, M., Haller, V., Steinmann, B., Lichtensteiger, W., 2001a. In vitro and in vivo estrogenicity of UV screens. Environ. Health Perspect. 109, 239; Schlumpf, M., Berger, L., Cotton, B., Conscience-Egli, M., Durrer, S., Fleischmann, I., Haller, V., Maerkel, K., Lichtensteiger, W., 2001b. Estrogen active UV screens. SÖFW-J. 7, 10]. 4-MBC (7, 24, 47mg/(kgday)) was administered in chow to long Evans rats from 10 weeks before mating of the parent (F0) generation until adulthood of the F1 generation. Peripheral reproductive organs and central nervous system were studied in adult offspring. mRNA expression of progesterone receptor (PR), an estrogen-regulated gene, was investigated in medial preoptic area (MPO) and ventromedial hypothalamic nucleus (VMH) by real-time RT-PCR. We analyzed intact 12-week-old male and female offspring under steady state conditions and adult gonadectomized offspring 6h after a single s.c. injection of estradiol-17ß (E2) (10 or 50µg/kg) in order to assess estrogen sensitivity. At steady state conditions we observed significantly higher PR mRNA expression in VMH of control females versus control males. 4-MBC exposed females exhibited a decrease in PR mRNA to levels of control males. The increase in PR mRNA in response to E2 was higher in VMH of males of both 4-MBC groups as compared to control males. PR mRNA levels were similar in MPO of control males and females. Developmental 4-MBC exposure increased PR mRNA levels in male MPO, but did not significantly change female levels. The acute response to the lower E2 dose was decreased in MPO of 4-MBC-exposed males, whereas females of the 7mg/kg dose group exhibited an increased reaction to 50µg/kg of E2. Our data indicate that developmental exposure to endocrine active chemicals such as the UV filter 4-MBC can interfere with sexually dimorphic gene expression in brain in a sex- and region-specific manner.

Differential gene expression patterns in developing sexually dimorphic rat brain regions exposed to antiandrogenic, estrogenic, or complex endocrine disruptor mixtures: glutamatergic synapses as target.

The study addressed the question whether gene expression patterns induced by different mixtures of endocrine disrupting chemicals (EDCs) administered in a higher dose range, corresponding to 450×, 200×, and 100× high-end human exposure levels, could be characterized in developing brain with respect to endocrine activity of mixture components, and which developmental processes were preferentially targeted. Three EDC mixtures, A-Mix (anti-androgenic mixture) with 8 antiandrogenic chemicals (di-n-butylphthalate, diethylhexylphthalate, vinclozolin, prochloraz, procymidone, linuron, epoxiconazole, and DDE), E-Mix (estrogenic mixture) with 4 estrogenic chemicals (bisphenol A, 4-methylbenzylidene camphor, 2-ethylhexyl 4-methoxycinnamate, and butylparaben), a complex mixture, AEP-Mix, containing the components of A-Mix and E-Mix plus paracetamol, and paracetamol alone, were administered by oral gavage to rat dams from gestation day 7 until weaning. General developmental endpoints were not affected by EDC mixtures or paracetamol. Gene expression was analyzed on postnatal day 6, during sexual brain differentiation, by exon microarray in medial preoptic area in the high-dose group, and by real-time RT-PCR in medial preoptic area and ventromedial hypothalamus in all dose groups. Expression patterns were mixture, sex, and region specific. Effects of the analgesic drug paracetamol, which exhibits antiandrogenic activity in peripheral systems, differed from those of A-Mix. All mixtures had a strong, mixture-specific impact on genes encoding for components of excitatory glutamatergic synapses and genes controlling migration and pathfinding of glutamatergic and GABAergic neurons, as well as genes linked with increased risk of autism spectrum disorders. Because development of glutamatergic synapses is regulated by sex steroids also in hippocampus, this may represent a general target of ECD mixtures.

Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals.

We evaluated and compared four in vitro assays to detect androgen agonists and antagonists in an international interlaboratory study. Laboratory 1 used a cell proliferation assay (assay 1) with human mammary carcinoma cells stably transfected with human androgen receptor. The other laboratories used reporter gene assays, two based on stably transfected human prostate carcinoma cells (assay 2) or human mammary carcinoma cells (assay 4), and the third based on transient transfection of Chinese hamster ovary cells (assay 3). Four laboratories received four coded compounds and two controls: two steroidal androgens, two antiandrogens, an androgenic control, 5alpha-dihydrotestosterone (DHT), and an antiandrogenic control, bicalutamide (ICI 176,334). All laboratories correctly detected the androgenic activity of 4-androsten-3,17-dione and 17alpha-methyltestosterone. For both compounds, the calculated androgenic potencies relative to the positive control (RAPs) remained within one order of magnitude. However, laboratory 3 calculated a 50-fold higher RAP for 4-androsten-3,17-dione. All assays detected and quantified the antiandrogenic effect of vinclozolin [median inhibitory concentration (IC50) values ranging from 1.1 times symbol 10(-7) M to 4.7 times symbol 10(-7) M]. In assays 2 and 3, vinclozolin showed partial androgenic activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50 values matched those of bicalutamide. Similarly, we found antiandrogenic activity for tris-(4-chlorophenyl)methanol. RAAP values were between 0.086 and 0.37. Three assays showed cytotoxicity for this compound at or above 1 times symbol 10(-5) M. In summary, all assays proved sensitive screening tools to detect and quantify androgen receptor-mediated androgenic and antiandrogenic effects of these chemicals accurately, with coefficients of variation between 8 and 90%.

UV filters with antagonistic action at androgen receptors in the MDA-kb2 cell transcriptional-activation assay.

The fact that certain ultraviolet (UV) filters used in cosmetics display estrogenic activity prompted us to study potential actions on androgen receptors (AR) in the human breast carcinoma cell line MDA-kb2, which expresses functional endogenous AR and glucocorticoid receptors (GR) and is stably transfected with a luciferase reporter plasmid. Dihydrotestosterone (DHT), methyltrienolone (R1881), methyltestosterone, danazol, and androstenedione increased luciferase activity, with EC50 values between 0.11 nM (R1881), 0.14 nM (DHT), and 73.5 nM (androstenedione). DHT-induced luciferase gene expression was inhibited by nonsteroidal antiandrogens, hydroxyflutamide, flutamide, bicalutamide, and vinclozolin. In contrast, the steroidal AR agonist/antagonist cyproterone actetate showed agonistic activity in the absence and presence of DHT, which was not blocked by hydroxyflutamide and thus seems not to be mediated by AR. GR-mediated activation of luciferase by dexamethasone was 100 times less potent than DHT and was not antagonized by hydroxyflutamide. The cell line was used for screening of UV filters, benzophenone-3 (Bp-3), benzophenone-4, 3-benzylidene camphor, 4-methylbenzylidene camphor, butyl-methoxy-dibenzoylmethane, homosalate (HMS), octyl-dimethyl-PABA, and octyl-methoxycinnamate. Two of these, Bp-3 and HMS, antagonized DHT-induced AR activation below cytotoxic concentrations, with IC50 of 5.57 10-6 M (HMS) and 4.98 10-6 M (Bp-3). None of the eight UV filters displayed agonistic activity when tested alone, but high concentrations of Bp-3 induced an increase of luciferase activity in the presence of dexamethasone, which was not blocked by hydroxyflutamide or the estrogen antagonist, ICI 182,780. These data indicate that the UV filters Bp-3 and HMS possess antiandrogenic activity in vitro in addition to estrogenic activity.

Estrogenic activity and estrogen receptor beta binding of the UV filter 3-benzylidene camphor. Comparison with 4-methylbenzylidene camphor.

UV filters represent new classes of estrogenic [Environ. Health Perspect. 109 (2001) 239] or antiandrogenic [Toxicol. Sci. 74 (2003) 43] chemicals. We tested 3-benzylidene camphor (3-BC), reported as estrogenic in fish [Pharmacol. Toxicol. 91 (2002) 204], and mammalian systems in comparison to 4-methylbenzylidene camphor (4-MBC), shown to be active in rats, and analyzed binding to estrogen receptor subtypes. 3-BC and 4-MBC stimulated MCF-7 cell proliferation (EC(50): 0.68 and 3.9 microM). The uterotrophic assay of 3-BC (oral gavage) in immature rats showed unexpected potency with ED50 45.3mg/kg per day; lowest effective dose 2mg/kg per day, and maximum effect with 70% of ethinylestradiol. After comparing with literature data, we found that the oral 3-BC was considerably more potent than oral bisphenol A and almost as active as subcutaneous genistein. 3-BC and 4-MBC displaced 16alpha 125I-estradiol from porcine uterine cytosolic receptors (IC(50): 14.5 and 112 microM), and from recombinant human estrogen receptor beta (hERbeta) (IC(50): 3-BC, 11.8 microM; 4-MBC, 35.3 microM), whereas no displacement was detected at human estrogen receptor alpha (hERalpha) up to 3mM. This subtype selectivity makes the two camphor derivatives interesting model compounds. Their activity on immature rat uterus is not easily explained by ERbeta activation. It cannot be excluded that active metabolites with possibly different receptor binding characteristics are formed in vivo.

Endocrine activity and developmental toxicity of cosmetic UV filters--an update.

UV filters represent a new class of endocrine active chemicals. In vitro, 8/9 chemicals showed estrogenic (MCF-7 cells), and 2/9 antiandrogenic activity (MDA-kb2 cells). Six/nine filters (benzophenone (Bp)-1, Bp-2, Bp-3, 3-benzylidene camphor (3-BC), 4-methylbenzylidene camphor (4-MBC), octyl-methoxycinnamate (OMC)) increased uterine weight in immature rats. 3-Benzylidene camphor and 4-MBC displaced 16alpha125I-estradiol from human estrogen receptor (ER)beta, not ERalpha. Developmental toxicity of 4-MBC (0.7-47 mg/kg body weight/day) and 3-BC (0.24-7 mg/kg), administered in chow was investigated in Long Evans (LE) rats. Weight gain of pregnant rats was reduced only by 3-BC, early postnatal survival rate and thymus weight by both compounds at higher doses. 4-Methylbenzylidene camphor and 3-BC delayed male puberty, and dose-dependently affected reproductive organ weights of adult male and female F1 offspring, with partly different effect patterns. Thyroid weight was increased by higher 4-MBC doses. Tissue-specific changes in mRNA levels of estrogen-regulated genes in prostate, uterus and brain regions, determined by real-time PCR, and in their response to acute estradiol challenge in adult gonadectomized offspring were observed. Lowest effective doses were 0.24 mg/kg/day for 3-BC and 7 mg/kg/day for 4-MBC. Fat tissue levels at 7 mg/kg 4-MBC (GC-MS) approached the range of UV filters in fish (Nagtegaal et al., 1997; Balmer et al., 2004).